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Astrocyte layers in the mammalian cerebral cortex revealed by a single-cell in situ transcriptomic map


Although the cerebral cortex is organized into six excitatory neuronal layers, it is unclear whether glial cells show distinct layering. In the present study, we developed a high-content pipeline, the large-area spatial transcriptomic (LaST) map, which can quantify single-cell gene expression in situ. Screening 46 candidate genes for astrocyte diversity across the mouse cortex, we identified superficial, mid and deep astrocyte identities in gradient layer patterns that were distinct from those of neurons. Astrocyte layer features, established in the early postnatal cortex, mostly persisted in adult mouse and human cortex. Single-cell RNA sequencing and spatial reconstruction analysis further confirmed the presence of astrocyte layers in the adult cortex. Satb2 and Reeler mutations that shifted neuronal post-mitotic development were sufficient to alter glial layering, indicating an instructive role for neuronal cues. Finally, astrocyte layer patterns diverged between mouse cortical regions. These findings indicate that excitatory neurons and astrocytes are organized into distinct lineage-associated laminae.

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Fig. 1: LaST map pipeline for mapping cortical neuronal subtypes in situ.
Fig. 2: Novel layer expression differences among cortical gray matter astrocytes revealed through RNA-seq and LaST map.
Fig. 3: Astrocytes show broad expression gradients across cortical depth and diverge from neuronal layers.
Fig. 4: Spatial reconstruction of astrocyte layers from single-cell transcriptome data.
Fig. 5: Evidence that post-mitotic neuronal cues establish astrocyte layer identities.
Fig. 6: Astrocyte arealization across the cortex.

Data availability

The raw bulk RNA-seq data are available at the Gene Expression Omnibus under the accession code GSE140822. The scRNA-seq data will be made available under Other data are available as Supplementary Materials or from the corresponding author upon request.

Code availability

The code for spatial reconstruction of single-cell astrocyte RNA-seq can be found at The SlideSegmenter code is available at The Harmony image analysis scripts are provided as Supplementary Materials. Other code is available upon request.


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We thank B. Barres and S. Teichmann for helpful discussions and comments. We thank B. Lynch and X.-J. Ma for advice on RNAScope, as well as R. Sawkins and J. Hutt for technical support on imaging. We also thank E. Olson (SUNY Upstate University) for providing Reeler mice and R. Marcucio (UCSF) for providing the Satb2-flox mice. The authors were supported by the Life Sciences Research Fellowship and the Howard Hughes Medical Institute (O.A.B.), the Wellcome Trust (T.B.), National Institute for Health Research (NIHR) Academic Clinical Fellowship and a Wellcome Trust PhD for Clinicians Fellowship (A.M.H.Y.) and Stichting Alzheiemer Onderzoek (A.M.). The present study was supported by the Paul G. Allen Foundation Distinguished Investigator Program (E.M.U. and D.H.R.), the Loulou Foundation, the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation (D.H.R., D.G. and G. C.), BRAIN initiative (grant no. 1U01 MH105991 to D.G.) and National Institutes of Health (grant nos. 1R01 MH109912 to D.G. and P01NS08351 to D.H.R.), NIHR and the European Union Seventh Framework (to P.H.), National Institute of Neurological Disorders and Stroke Informatics Center for Neurogenetics and Neurogenomics (grant no. P30 NS062691 to G.C.), Wellcome Trust core support (M.H. and O.A.B.), European Research Council (grant no. 281961 to M.G.H.), Fonds Wetenschappelijk Onderzoek (grant nos. G066715N and 1523014N to M.G.H.), Stichting Alzheimer Onderzoek (S no. 16025 to M.G.H.) and VIB Institutional Support and Tech Watch funding (to M.G.H.), Howard Hughes Medical Institute and the Wellcome Trust (to D.H.R.).

Author information




O.A.B. and D.H.R. conceived the study. O.A.B. planned the experiments. O.A.B., T.B., S.H. and K.R. performed histology and imaging experiments. O.A.B. performed the image analysis. K.P. and J.S. contributed to the histology and imaging pipeline. D.P. and O.A.B. analyzed neuron gene expression data to identify subtypes. A.B. wrote the SlideSegmenter software. A.M.H.Y., P.H., M.F.P. and E.H. provided human tissue. O.A.B. and L.B.H. performed layer astrocyte purification. O.A.B. and R.K. analyzed RNA-seq data. G.S., D.H.G. and E.M.U. supervised analysis of neuron smFISH and astrocyte RNA-seq data. A.M., M.B. and M.G.H. generated the astrocyte scRNA-seq data. V.K. and M.H. analyzed the scRNA-seq data and performed spatial reconstruction analysis. K.S. and S.C. supported mouse work and genotyping. O.A.B. and D.H.R. wrote the manuscript with feedback from all authors.

Corresponding authors

Correspondence to Omer Ali Bayraktar or David H. Rowitch.

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The authors declare no competing interests.

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Peer review information Nature Neuroscience thanks F. Guillemot, N. Sestan and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary information

Supplementary Information

Supplementary Figs. 1–22 and Supplementary Tables 6 and 7.

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Supplementary Table

Supplementary Tables 1–5 and 8–11.

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Bayraktar, O.A., Bartels, T., Holmqvist, S. et al. Astrocyte layers in the mammalian cerebral cortex revealed by a single-cell in situ transcriptomic map. Nat Neurosci 23, 500–509 (2020).

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